To understand and control disease, it is critical to focus on proteins. Proteins consist of their peptide backbone with post-translational modifications (PTMs), the latter of which control the function of the protein. The goal of this research continues to be the development of tools to comprehensively characterize proteins immunoextracted from cells, tissues, or biological matrices at the fmole level. We propose to develop a general platform involving LC/MS and CE-LIF-MS for full glycosylation characterization, including glycan linkage and position isomers at individual sites of multisite glycosylated proteins. All forms on specific sites above the ~10% level will be quantitated. With this information, we will then focus on trace quantitation of multiple forms on multiple sites by multiple reaction monitoring (MRM) LC/MS using our highly sensitive 10 5m i.d. porous layer open tubular (PLOT) LC columns. As a demonstration of the power of the technology, we will immunoprecipitate glycoproteins from small volumes of plasma, and quantitate a number of specific glycoforms on specific sites of healthy individuals and those with breast cancer and other diseases. This technology will allow us to monitor the variation in glycan structures and the relative levels of individual forms on specific sites of glycoproteins for disease vs. control, as well glycan variability within each cohort. A second demonstration will involve full determination of the extracellular domain of the important receptor tyrosine kinases - EGFR and Her2. We will conduct a variety of studies with cell lines for EGFR alone and EGFR/Her2, comprehensively characterizing glycosylation and the dynamics of phosphorylation as a function of stimulation. These results will be compared to those obtained when hypoxia conditions (low oxygen levels) are used for cell growth, as would occur in tissue. Finally, we will demonstrate comprehsive characterization of EGFR extracted from mouse and human tissue. The research will result in powerful new tools to allow a deeper understanding of biological processes and disease.